1. Field of the Invention
The present invention relates to an isolated cellular composition comprising a substantially homogeneous population of mammalian neuronal progenitor cells. Additionally, the present invention relates to methods of delivering biologically active molecules to a mammalian brain by transplanting the cellular composition to the brain.
2. Background Art
Because mammalian neurons are generally incapable of dividing when mature, sources of dividing neuronal cells have been sought. Several difficulties have arisen, however, in identifying sources of dividing cells that generate neurons because neuronal progenitor cells frequently fail to express neuronal markers and because heterogeneous populations of cells (including neuronal and non-neuronal cells) generally arise.
Neoplastic cell lines and immortalized neuronal precursors have been used to provide relatively homogeneous populations of cells. Because these cells are rapidly dividing, they generally show a limited ability to fully differentiate into cells with a neuronal phenotype. For example, PC12 cells derived from a pheochromocytoma fail to differentiate or maintain a differentiated state in culture in the absence of nerve growth factor (NGF). (Green and Tischler, Advances in Cellular Neurobiology, S. Federoff and L. Hertz, eds. (Academic Press, N.Y.), (1982). Additionally, these cells are tumor-derived and have neoplastic characteristics.
Similarly, embryonal carcinoma cell lines have been differentiated in culture under special conditions. NT2 cells, derived from a teratocarcinoma, will differentiate in culture only following extended treatment with retinoic acid. The NT2 cells, however, differentiate into both neuronal and non-neuronal cell types. The resulting mixed culture must be treated with mitotic inhibitors and then the cells replated to remove the dividing non-neuronal cells and approach a relatively pure population of neuronal cells. (U.S. Pat. No. 5,175,103). These relatively pure neuronal cells nonetheless are tumor-derived and have neoplastic characteristics.
Sources of neuronal precursors from adult and neonatal mammalian nervous systems have generally resulted in similar problems with heterogeneity. Reynolds and Weiss, Science 255:1707 (1992), have cultured cells from the adult striatum, presumably including portions of the subventricular zone. The cells were cultured in the presence of epidermal growth factor (EGF) and allowed to form large cell clusters, which were termed "neurospheres." The spheres were then dissociated and the cells were cultured in the presence of EGF. The resulting cell cultures consisted of a mixture of post-mitotic neurons, glia, and subependymal cells. Thus, by these means, some of the newly-generated cells were induced to differentiate into neurons; however, the proportion of neurons obtained is low by this method. Others have been able to induce some neuronal proliferation from cultures of the neonatal telencephalon, by administration of fibroblast growth factor. Like the method of Reynolds and Weiss, this neonatal source also results in low proportions of neurons compared to non-neuronal cells. Relatively pure populations of neuronal cells can be achieved by these methods only following treatment with mitotic inhibitors. Therefore, the relatively pure neuronal cells are post-mitotic.
The subventricular zone is known to be a source of certain dividing cells in the nervous system. However, the subventricular zone has been viewed exclusively as a source of glia and not neurons (Paterson et al., J Comp. Neurol., 149:83, 1973; LeVine and Goldman, J Neurosci, 8:3992, 1988; Levison and Goldman, Neuron 10:201 (1993). This was the consensus concerning the intact, in vivo subventricular zone. Luskin, Neuron, 11:173 (1993) found that a discrete region of the intact subventricular zone produced numerous neurons that differentiated into olfactory bulb neurons in vivo. However, investigators who have cultured cells derived from the neonatal subventricular zone have shown that the vast majority of these cells become glia when cultured (Vaysse and Goldman, Neuron, 4:833, 1990; Lubetzki et al., Glia, 6:289, 1992). Lois and Alvarez-Buylla, Proc. Natl. Acad Sci., 90:2074, (1993) cultured explants of the subventricular zone from adult mammalian forebrain, and found a preponderance of glia.
Thus, a simple means of obtaining a composition of cells having a high percentage of neuronal progenitor cells and a correspondingly low percentage of non-neuronal cells is needed. Such a composition and method for achieving the composition would offer several advantages over prior compositions and methods. Dividing cells can be manipulated through gene transfer. In addition, neuronal cells which differentiate and eventually cease dividing result in a decreased likelihood of tumor formation when transplanted into a host nervous system. Glia, in contrast to neurons, can be highly proliferative when given certain signals and can even form gliomas. Neoplastic cell lines can similarly result in tumor formation.
In contrast to the above-described studies which support that only glia arose from the cultured telencephalic subventricular zone or that only a low fraction of neurons could be obtained under particularly favorable conditions, the present invention provides an isolated cellular composition comprised of a substantially homogeneous population of mammalian, non tumor-derived neuronal progenitor cells which express a neuron-specific marker and which can give rise to progeny which can differentiate into neuronal cells. This ability of these cells to divide is atypical because most cells expressing neuron-specific cell markers are post-mitotic cells. Also, the present composition comprises a population of cells of such homogeneity that greater than about 90% of the neuronal progenitor cells express a neuron-specific marker and can give rise to progeny which can differentiate into neuronal cells.